Patient samples and ethical statement
This study enrolled 50 patients with primary GC from January 2016 to December 2018 at the Department of General Surgery, Changzheng Hospital, Navy Medical University, Shanghai, China. All patients were pathologically diagnosed with GC in accordance with the American Joint Committee on Cancer criteria. No patients had received preoperative chemotherapy or radiotherapy. The gastric tissues were obtained following Institutional Review Board-approved protocols. This study was also in accordance with the Declaration of Helsinki. Informed consent was provided by all enrolled patients. ENCORI (https://starbase.sysu.edu.cn/), an interactive web server, was used to compare the expression levels of SOD2 in TCGA-STAD cohort with adjacent normal TCGA samples. The GSE60427 dataset included 8 normal and 24 H. pylori-positive specimens from gastric mucosal biopsies and was obtained from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). The animal experiments were approved by the Institutional Animal Care and Use Committee of Changzheng Hospital, Navy Medical University, in accordance with the Guide for the U.S. Public Health Service’s policy on laboratory animals.
Cell Culture And Reagents
Human GC cell lines AGS, MGC803, and BGC823 were obtained from ATCC (Manassas, VA, USA). The HGC-27, MKN45, and GES-1 cell lines were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The cell lines were authenticated by short tandem repeat-based assay. AGS, BGC823, MKN45, and HGC-27 were cultured at 37°C in a humidified atmosphere containing 5% CO2 with RPMI-1640 medium with 10% FBS. MGC-803 and GES-1 cells were maintained at 37°C in a humidified atmosphere of 5% CO2 with Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS. Recombinant human TNF-α (#300-01A, PeproTech, NJ, USA), 5-fluorouracil (5-FU) (#ST1060, Beyotime, Shanghai, China), and NF-κB inhibitor Bay 11–7082 (#S2913, Selleckchem, Houston, TX, USA) were purchased. Erastin (#abs810744) and ferrostatin-1 (#abs813072) were obtained from univ-bio (Shanghai, China). The final concentrations used in the experiments were as follows: ferrostatin-1, 2 µM; and erastin, 20 µM.
Immunohistochemistry (IHC)
Paraffin-embedded clinical samples were used for immunostaining to detect SOD2 protein expression. In brief, tumor tissues were fixed with 4% (v/v) formaldehyde, embedded in paraffin, and cut into 5 µm thick sections. The sections were incubated with the primary antibody against SOD2 (1:500; #13141S, Cell Signaling Technology) overnight at 4°C in a moist chamber. The sections were incubated with HRP-conjugated secondary antibody for 15 min at room temperature, and then stained with DAB and hematoxylin. The IHC results were scanned by whole-slide scanning Pannoramic scanner (3D HISTECH, Waltham, MA, USA). The protein expression levels were tested and scored based on intensity and frequency (Zhu et al. 2017).
H. pylori culture and infection
Wild-type Cag+ H. pylori strains 26695, 43504, and the rodent-adapted PMSS1 were obtained from ATCC. Briefly, H. pylori strains were maintained in brain heart infusion agar (OXOID, Thermo Fisher Scientific, USA) with 10% sheep blood. Subsequently, the strains were cultured at 37°C and placed in an incubator with 85% N2, 5% O2, and 10% CO2 for 16 hours. H. pylori were co-cultured with GC cells at a multiplicity of infection (MOI) of 100.
RNA Sequencing Analysis (RNA-seq)
Next-generation RNA sequencing analysis was applied to test the mRNA expression features by Illumina HiSeq 4000 (Illumina, San Diego, CA, USA). Significant differential expression of a gene was identified as a > 2-fold difference between treatment and control samples with a P value < 0.05. The heat map was visualized by Gene Ontology (GO) using Cluster software. Differentially expressed genes (DEGs) were analyzed using GO analysis. Finally, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations were performed to analyze the enrichment of DEGs.
RNA Interference, Plasmid Construction, And Transfection
The SOD2 siRNAs (GenePharma Corporation, Shanghai, China) at a final concentration of 100 nmol/L were transfected into GC cells using the Lipofectamine™ 3000 transfection kit (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions. The SOD2 siRNA1 sense was 5′-GTGGTGGTCATATCAATCATT-3′ and SOD2 siRNA2 sense was 5′- GTGGTGGTCATATCAATCATT-3′. The coding sequence of SOD2 was cloned in pcDNA3.1 plasmid (Invitrogen). Human SOD2 overexpression plasmids and control plasmids were transfected into GC cells by Lipofectamine™ 3000 reagent in accordance with the manufacturer’s instructions.
Western Blot
Cells were washed with pre-cooled PBS and lysed using RIPA buffer. Lysates were then centrifuged at 13000 rpm for 10 min at 4°C. Briefly, proteins were separated by 10% SDS-PAGE and transferred to a PVDF membrane (Millipore). We then incubated the blots with primary antibodies specific to SOD2 (1:1000; #13141S, Cell Signaling Technology), CagA (1:300; #sc-28368, Santa Cruz Biotechnology), P65 (1:1000; #8242S, Cell Signaling Technology), p-P65 (1:1000; #3033S, Cell Signaling Technology), AKT (1:1000; #4685S, Cell Signaling Technology), p-AKT (1:1000; #4060S, Cell Signaling Technology), 4-hydroxy-2-nonenal (4-HNE) (1:1000; #ab46545, Abcam), or GPX4 (1:1000; #67763-1-Ig, Proteintech) overnight at 4°C, and then a secondary antibody according to the host specificity for 1 h. GAPDH (1:1000; #5174S, Cell Signaling Technology) was used as the loading control. The blot analysis was visualized by the Clarity™ Western ECL Substrate (Bio-Rad, #1705060).
Quantitative Real-time Pcr
Isolation of total RNA was performed using TRIzol reagent (Ambion, # 15596018). Total RNA (1 µg) was reverse transcribed by an iScript™ cDNA synthesis kit (Bio-Rad, CA, USA). cDNA was used to analyze the mRNA levels of SOD2 by qRT-PCR assay using iTaq Universal SYBR Green Supermix (Bio-Rad). The primers for human SOD2 were: 5′-GCTGGAAGCCATCAAACGTG-3′ (forward) and 5′-GCAGTGGAATAAGGCCTGTTG-3′ (reverse). Primers for GPX4 were 5′-CCGATACGCTGAGTGTGGTT-3′ (forward) and 5′-TGGAGAGACGGTGTCCAAAC-3′ (reverse). Primers for GAPDH were 5′-GGACCTGACCTGCCGTCTAG-3′ (forward) and 5′-GTAGCCCAGGATGCCCTTGA-3′ (reverse). Each reaction was performed in triplicate. The relative expression levels of genes were analyzed by the 2−ΔΔCt method. GAPDH was used as a normalization control.
Cell Viability Assay
Ten thousand cells were seeded in a 96-well plate and treated with erastin, ferrostatin-1, or dimethylsulfoxide (DMSO) for 12 h. Then, 100 µL of medium containing 10% CCK-8 reagent was added in accordance with the manufacturer’s instructions (Dojindo, Kumamoto, Japan). Each group had three duplicate wells. Following incubation at 37°C for 1 h, the absorbance was then measured using a Safire2 microplate reader (Tecan, Switzerland) at 450 nm.
For cell viability analysis, 106 GC cells/well were seeded on six-well plates. Cells were treated with erastin, ferrostatin-1, or DMSO for 12 h. The trypan blue exclusion staining assay was then used to evaluate the cell viability. Cell viability was detected by a cell viability analyzer (Beckman). All the experiments were conducted in triplicate.
Superoxide Assay
The cellular superoxide contents were examined by a superoxide assay kit (Beyotime, Shanghai, China) following the referenced protocol (Mo et al. 2019). GC cells with different treatments were incubated with the superoxide detection reagents for 30 min at room temperature. Subsequently, the absorbance was detected by a spectrophotometer (Thermo Fisher Scientific) (OD = 450 nm).
ROS assay
As previously described (Esteban et al. 2010), ROS concentrations were tested by a Reactive Oxygen Species Assay Kit (Beyotime, Shanghai, China). Following the applied treatment, GC cells were co-incubated with DCFH-DA for 30 min at 37°C and subsequently detected by fluorescence spectrophotometer (Thermo Fisher Scientific).
Lipid Peroxidation Assay
The malonic dialdehyde (MDA) concentrations of cell lysates were evaluated by Lipid Peroxidation Assay Kits (Beyotime, Shanghai, China) in accordance with the manufacturer’s instructions. The assay detects the MDA-thiobarbituric acid (TBA) adducts generated by the reaction of MDA with TBA. The MDA-TBA adducts can be further measured using colorimetry.
Luciferase Reporter Assay
The specific sequences in the SOD2 promoter were acquired from the National Center for Biotechnology Information, and then subcloned into pGL3-Basic (Promega, Madison, WI, USA) to construct the luciferase reporter plasmids (P1: 1200-0 bp upstream of the SOD2 coding sequence, P2: 1000-0 bp upstream of the SOD2 coding sequence, P3: 600-0 bp upstream of the SOD2 coding sequence). For luciferase analysis, 293T cells were seeded in a 12-well plate and co-transfected with NF-κB expression vector and the luciferase plasmids for 24 h. Subsequently, cells were analyzed using a Dual-Luciferase Assay following the manufacturer’s instructions (Promega). Each transfection was performed in triplicate.
Chromatin Immunoprecipitation Assay
MGC803 cells were crosslinked with 1% formaldehyde and resuspended in 400 µl lysis buffer. The crosslinked DNA was then sonicated into 200–500 bp fragments for the following DNA pull-down. The antibodies used for chromatin immunoprecipitation (ChIP) were as follows: NF-kB (Ser536) (#3033S, Cell Signaling Technology), mouse IgG (Millipore Merck), and protein A/G magnetic beads (#CS204457, Millipore Merck). These were added to the cell lysates and incubated at 4°C overnight. Elution of the immunoprecipitated chromatin complexes was followed by DNA purification, and the purified DNA was further analyzed by PCR assay. We designed the following ChIP primers for the NF-κB binding sites: SOD2-1-F: 5′-AGCAAAGGTCAAGGTGGGTG-3′, SOD2-1-R: 5′-GTCTCCTTCGTGCCGCATGGT-3′; SOD2-2-F: 5′-ATGACAGTGGAGCCCAGGTCCT-3′, SOD2-2-R: 5′-TGTAGGAACGTGTGGGAGGA-3′; SOD2-3-F: 5′-CACAGGCCATCATACCTGGC-3′, SOD2-3-R: 5′-AGCCCTGTCAGTAGCTTCTA-3′.
Clonogenic Assay
Approximately 300 GC cells were seeded on six-well plates, infected with H. pylori, and cultured at 37°C and 5% CO2 for 2 weeks. Briefly, GC cells were fixed with paraformaldehyde and dyed with 0.2% (w/v) crystal violet. Subsequently, images were acquired and the colonies were counted.
In vivo experiments
Mice were housed in a specific-pathogen-free animal laboratory in 12 h light–dark cycles. Twenty 6-week-old male C57BL/6 mice (SLAC LABORATORY ANIMAL, Shanghai, China) were randomly assigned into the control and PMSS1 groups with 10 mice per group. H. pylori strain PMSS1 (109 CFU/mouse) was used for orogastric gavage, while the control mice were treated with Brucella broth. Two weeks later, mice were euthanized, and the gastric tissues were used for western blot and qRT-PCR analysis.
Statistical analysis
All data presented are representative of at least three independent experiments and expressed as the mean ± standard deviation (SD). The differences between groups were analyzed by one-way analysis of variance (ANOVA) and Student’s t-test. Overall survival (OS) was analyzed with the Kaplan–Meier method. All statistical analyses were conducted using SPSS 16.0 and GraphPad Prism 8.0. A two-tailed value of P < 0.05 was considered to be statistically significant.